An interdisciplinary team of biological, chemical, and physical scientists from Stanford University and Kent State University will embark upon a high-risk/high-potential-payoff program to design, develop and apply unique fluorophores to explore protein localization in cells, down to the single-molecule level. This will involve (i) design, synthesis, characterization and optimization of a new class of fluorescent tags for biological imaging, (ii) development of innovative cellular targeting technologies for these new fluorophores to determine location and co-location of proteins, and (iii) demonstration of methods for detection of coordinated of protein location (or mislocation in mutant strains) and gene expression to explore the regulatory function of spatial positioning in bacteria. Item (iii) is particularly important, in that the new fluorophores and targeting protocols will be challenged by a specific need and will be tested directly on a key model biological system. The members of the research team have recently demonstrated optical detection of fluorescent single molecules in living cells using both extrinsic fluorescent antigenic peptides as well as green fluorescent protein mutant fusions. In this application, the new fluorophores to be designed, synthesized, and applied are based on a class of fluorophores with exceptional fluorescence properties which already allow detection at the single-copy level. As single-molecule labels, these molecules provide more than an order of magnitude improvement in photophysical properties compared to green fluorescent protein mutants, thus the ability to detect single molecules in cells should be improved by more than an order of magnitude, opening a new frontier in the observation of dynamical events in living cells. In addition to single-molecule experiments, the new reporter fluorophores generated by this project will enable advances in time-lapse fluorescence microscopy by virtue of increased signal-to-noise and photobleaching resistance, properties that will allow lower concentrations of labels to be used in cellular fluorescence investigations.